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首页> 外文期刊>American Journal of Physiology >Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice.
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Isolation and characterization of coronary endothelial and smooth muscle cells from A1 adenosine receptor-knockout mice.

机译：A1腺苷受体敲除小鼠的冠状动脉内皮和平滑肌细胞的分离和鉴定。

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Mice have been used widely in in vivo and in vitro cardiovascular research. The availability of knockout mice provides further clues to the physiological significance of specific receptor subtypes. Adenosine A(1) receptor (A(1)AR)-knockout (A(1)KO) mice and their wild-type (A(1)WT) controls were employed in this investigation. The heart and aortic arch were carefully removed and retroinfused with enzyme solution (1 mg/ml collagenase type I, 0.5 mg/ml soybean trypsin inhibitor, 3% BSA, and 2% antibiotics) through the aortic arch. The efflux was collected at 30-, 60-, and 90-min intervals. The cells were centrifuged, and the pellets were mixed with medium [medium 199-F-12 medium with 10% FBS and 2% antibiotics (for endothelial cells) and advanced DMEM with 10% FBS, 10% mouse serum, 2% GlutaMax, and 2% antibiotics (for smooth muscle cells)] and plated. Endothelial cells were characterized by a cobblestone appearance and positive staining with acetylated LDL labeled with 1,1'-dioctadecyl-3,3,3',3-tetramethylindocarbocyanine perchlorate. Smooth muscle cells were characterized by positive staining of smooth muscle alpha-actin and smooth muscle myosin heavy chain. Homogeneity of the smooth muscle cells was approximately 91%. Western blot analysis showed expression of smoothelin in the cells from passages 3, 7, and 11 in A(1)WT and A(1)KO mice. Furthermore, the A(1)AR was characterized by Western blot analysis using an A(1)AR-specific antibody. To our knowledge, this is the first isolation and successful characterization of smooth muscle cells from the mouse coronary system.
机译：小鼠已广泛用于体内和体外心血管研究。基因敲除小鼠的可用性为特定受体亚型的生理意义提供了进一步的线索。腺苷A（1）受体（A（1）AR）敲除（A（1）KO）小鼠和他们的野生型（A（1）WT）控件用于这项研究。小心地取出心脏和主动脉弓，并通过主动脉弓向后注入酶溶液（1 mg / ml I型胶原酶，0.5 mg / ml大豆胰蛋白酶抑制剂，3％BSA和2％抗生素）。每隔30分钟，60分钟和90分钟收集一次流出。将细胞离心，然后将沉淀与培养基[含10％FBS和2％抗生素的199-F-12培养基（用于内皮细胞）和含10％FBS，10％小鼠血清，2％GlutaMax的高级DMEM混合，和2％的抗生素（用于平滑肌细胞）]并镀上。内皮细胞的特征是鹅卵石外观，并用被1,1'-二十八烷基-3,3,3'，3-四甲基吲哚碳花青高氯酸盐标记的乙酰化LDL染色呈阳性。平滑肌细胞的特征在于平滑肌α-肌动蛋白和平滑肌肌球蛋白重链的阳性染色。平滑肌细胞的均质性约为91％。蛋白质印迹分析显示，在A（1）WT和A（1）KO小鼠的第3、7和11代中，细胞中的平滑肌表达。此外，通过使用A（1）AR特异性抗体的蛋白质印迹分析来表征A（1）AR。据我们所知，这是首次从小鼠冠状动脉系统中分离并成功表征了平滑肌细胞。

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